Sox9基因诱导脂肪干细胞向软骨细胞分化

目的:观察Sox9基因对人脂肪干细胞(ADSCs)的诱导作用.方法:分离、纯化、培养人源ADSCs,并绘制生长曲线,传代三次后的ADSCs利用脂质体转染Sox9基因,选用抗生素G418进行筛选.以空载体转染细胞作对照,分别取48h和14d转染后的细胞做Flag蛋白免疫组织化学鉴定.通过检测转染细胞中Ⅱ型胶原来确定ADSCs是否向软骨细胞分化.结果:ADSCs呈长梭形,形态与骨髓间充质干细胞相似,600μ/ml G418为最适筛选浓度.转染后第48h和14d的细胞均能表达Sox9基因融合表达的Flag蛋白.第48h和14d,转染效率分别为93%和75%.转染后14d的ADSCs表达Ⅱ型胶原,转染后48h实验组和对照组都为阴性.结论:Sox9基因能诱导脂肪干细胞向软骨细胞分化.     

起搏基因体外调控骨髓间充质干细胞分化为类起搏细胞的研究

目的本研究旨在通过体外构建起搏基因质粒pIRES2-EGFP-HCN2,电穿孔转染骨髓间充质干细胞,检测其在体外的表达情况。方法对含mHCN2 cDNA的PTR载体进行转化和扩增,将所得mHCN2基因定向克隆到含有增强型绿色荧光蛋白的真核表达载体pIRES2-EGFP中,进行双酶切来鉴定克隆的正确性。将重组质粒及空白质粒用电穿孔法转染骨髓间充质干细胞,并在体外与心肌细胞共培养,观察搏动频率变化及mHCN2的表达,并检测其电生理和组织学特征。结果构建了重组质粒pIRES2-EGFP-HCN2。荧光显微镜下可见转染后的骨髓间充质干细胞呈绿色荧光,细胞中mHCN2的阳性表达率为98.2%。免疫荧光显示转染起搏基因的骨髓间充质干细胞mHCN2的表达,而对照组无表达。实验组共培养的心肌细胞搏动频率较对照组干细胞共培养的明显增快(140±11次/分VS 100±13次/分,P0.05),动作电位显示实验组最大舒张期电位值小于对照组(-62±2mv VS-71±2mv,P0.05)。免疫荧光显示干细胞与心肌细胞间形成间隙连接。结论成功构建了重组质粒pIRES2-EGFP-HCN2,转染后的骨髓间充质干细胞可在体外成功表达功能性mH-CN2通道,提供起搏电流,具有类起搏细胞的功能,为进一步构建生物起搏器提供依据。     

人HCN2 真核表达载体的构建及在HEK293 细胞中的表达

目的:构建含有人HCN2基因的真核表达载体,并观察在人胚胎肾细胞(HEK293)中的表达情况。方法:对人HCN2基因全序列进行分析,进行oligo设计,通过PCR,扩增HCN2全长cDNA,通过双酶切(XhoI和BamHI)装入真核表达载体pIRES2-EGFP中,脂质体法转染入HEK293细胞中,利用真核表达载体中带有绿色荧光蛋白GFP报告基因,对转染效率进行监测,采用反转录-聚合酶链反应检测HCN2 mRNA表达,全细胞膜片钳技术检测HCN2通道电流。结果:测序及酶切结果表明HCN2基因正确,荧光显微镜下,转染细胞观察到绿色荧光,反转录-聚合酶链反应检测到HCN2 mRNA表达,膜片钳检测到hHCN2基因编码的通道电流。结论:成功地构建了HCN2真核表达载体并进行了起搏通道HCN2基因的异源性表达。     

人HCN2真核表达载体的构建及在HEK293细胞中的表达

目的：构建含有人HCN2基因的真核表达载体,并观察在人胚胎肾细胞（HEK293）中的表达情况。方法：对人HCN2基因全序列进行分析,进行oligo设计,通过PCR,扩增HCN2全长cDNA,通过双酶切（XhoI和BamHI）装入真核表达载体pIRES2-EGFP中,脂质体法转染入HEK293细胞中,利用真核表达载体中带有绿色荧光蛋白GFP报告基因,对转染效率进行监测,采用反转录-聚合酶链反应检测HCN2 mRNA表达,全细胞膜片钳技术检测HCN2通道电流。结果：测序及酶切结果表明HCN2基因正确,荧光显微镜下,转染细胞观察到绿色荧光,反转录-聚合酶链反应检测到HCN2 mRNA表达,膜片钳检测到hHCN2基因编码的通道电流。结论：成功地构建了HCN2真核表达载体并进行了起搏通道HCN2基因的异源性表达。     

TGF-β1基因转染大鼠脂肪干细胞向软骨细胞分化的实验研究

目的TGF-β1基因转染大鼠的ADSCs(adipose stromal cells,ADSCs),诱导其向软骨细胞分化,为软骨组织工程学种子细胞提供新方法。方法分离、培养大鼠的ADSCs,免疫荧光法进行鉴定;TGF-β1基因转染ADSCs,对转染后细胞进行筛选;MTT法测定筛选后细胞的增殖活性;RT-PCR、Western blot对筛选后细胞的表达进行检测。结果成功分离、培养大鼠的ADSCs;细胞表面标志物CD29和CD44表达阳性,CD106和CD34表达阴性;基因转染后细胞的增殖力变强;转染TGF-β1的ADSCs在目的基因TGF-β1、SOX9、Aggrecan、Collagen II mRNA的表达和软骨特异性基质-Collagen II的分泌增强,明显高于对照组和转染空载体组;结果TGF-β1基因转染后ADSCs具有了软骨细胞的表型特征,可以用作软骨组织工程的种子细胞。     

Nurr-1基因转染促进脂肪干细胞的神经分化

目的:研究孤儿核受体相关基因1(Nurr-1)对脂肪干细胞(adipose tissue-derived stem cells,ADSC)向神经元方向分化的潜在作用。方法:流式细胞术与成骨、成脂诱导技术鉴定脂肪干细胞;Nurrr-1基因转染脂肪干细胞后,应用神经特异性标志物MAP-2,β-tubulin的免疫荧光染色评估其向神经方向分化的能力。结果:流式细胞术结果表明培养的细胞CD29,CD44表达90%以上,CD45,CD90表达均低于1.5%,经过诱导后,油红O、茜素红S染色均呈阳性,表明所培养的细胞为脂肪干细胞;慢病毒转染Nurr-1基因后,免疫荧光染色检测MAP-2,β-tubulin的免疫荧光强度显著增加;RT-PCR结果显示Nurr-1转染的脂肪干细胞的MAP-2、β-tubulin、NF200的表达量显著提高。结论:Nurr-1基因转染能促进脂肪干细胞向神经方向分化,为神经损伤和神经退行性病变的治疗提供了新途径。     

重组腺病毒Ad5-ADRbeta2-EGFP转染骨髓间充质干细胞移植治疗心衰大鼠的实验研究

目的:重组腺病毒Ad5-ADRbeta2-EGFP转染骨髓间充质干细胞(BMSCs)并植入心衰大鼠心肌,观察其心功能的变化.方法:采用部分缩窄腹主动脉法制备大鼠压力超负荷性慢性心力衰竭模型.分离和培养成年大鼠BMSCs,待细胞传代到第6代,用重组腺病毒Ad5-ADRbeta2-EGFP转染BMSCs48h后将BMSCs移植入心衰大鼠心肌内.实验共分3组:心衰组、移植BMSCs组及移植转染Ad5-ADRbeta2-EGFP的BMSCs组.移植BMSCs(1×104)4周后,检测大鼠血流动力学各指标的变化.结果:荧光显微镜下可见转染BMSCs绿色荧光蛋白表达达到80%以上.移植BMSCs组和移植转染Ad5-ADRbeta2-EGFP的BMSCs组的左心室舒张末压力(LVEDP)低于心衰组,左心室内压变化最大速率(±dp/dt max)和心率均高于心衰组,差异均有统计学意义(P<0.05).移植转染Ad5-ADRbeta2-EGFP的BMSCs组的左心室内压上升最大速率(+dp/dt)高于移植BMSCs组,差异有统计学意义(P<0.05).结论:重组腺病毒Ad5-ADRbeta2-EGFP转染BMSCs并移植入心衰大鼠后,能够改善心衰大鼠的心功能.     

脂质体介导的人甲状腺过氧化物酶基因转染肺癌A549细胞及其蛋白表达

目的:利用基因转染技术,研究以脂质体Lipofectamine2000为载体介导的人甲状腺过氧化物酶(TPO)基因体外转染肺癌细胞,检测感染细胞内TPO蛋白的表达,为放射性碘治疗肺癌提供理论和实验依据.方法:将获得的含TPO基因的质粒pcDAN3.1-hTPO进行扩增、纯化,并经酶切鉴定和DNA测序.将肺癌A549细胞在体外复苏与培养并分为两组:转染质粒peDAN3.1-hTPO的为实验组,转染空质粒pcDAN3.1的为对照组.以脂质体Lipofectamine2000为栽体,介导TPO基因转染肺癌细胞.采用Western Blot免疫印迹法和免疫组化法分别检测肺癌细胞中TPO蛋白的表达.结果:①酶切鉴定和DNA测序结果表明质粒pcDAN3.1-hTPO中插入的基因为hTPO基因,其片段大小和方向正确.②体外培养的肺癌细胞活力及数量正常,细胞活力为96%,细胞生长密度为1× 106/ml,满足实验要求.③质粒转染A549细胞后,Western Blot免疫印迹法显示:在实验组中,肺癌A549细胞有TPO蛋白的表达,而在对照组中无表达.④免疫组化染色结果显示:在实验组的肺癌A549细胞中,TPO蛋白表达阳性且主要分布于细胞膜上,阳性表达率可达75%,而在对照组中TPO蛋白表达阴性,两组比较差异有显著性(P=0.000).结论:①获得的hTPO基因片段的核苷酸序列与GeneBank报道完全一致.②在脂质体Lipofectamine2000的介导下,TPO基因能够有效地转染肺癌细胞.③转染人甲状腺过氧化物酶基因的肺癌细胞能够在体外成功地表达TPO蛋白.     

人HCN4通道的滞后现象:影响窦房结起搏的潜在决定因素(英文)

超极化活化环核苷酸门控(hyperpolarization-activated cyclic-nucleotide-gated,HCN)通道参与调制心脏跳动的节律和速率。与HCN1和HCN2有所不同,慢通道HCN4可能不存在电压依赖的滞后现象。本研究采用单细胞膜片钳方法,在稳定转染hHCN4的HEK293细胞上进行电生理记录,观察hHCN4通道是否存在滞后现象,以及cAMP对其的调制作用;同时采用实时定量RT-PCR方法检测窦房结和心房组织中HCNs的表达。电压钳实验结果显示hHCN4电流(Ih)激活随着保持电位超极化的变化而向去极化方向移动。三角电位变化钳(triangular ramp)和动作电位钳的结果也显示了hHCN4的滞后现象。cAMP增加Ih电流幅度,且使电流激活向去极化方向移动,从而改变内源性hHCN4滞后行为。RT-PCR结果显示,人窦房结组织主要表达HCN4,占75%,HCN1占21%,HCN2占3%,HCN3占0.7%。以上结果提示,人窦房结组织主要表达HCN4亚型,hHCN4的Ih存在电压依赖性的滞后现象,且受cAMP调制。由此推断,hHCN4通道的滞后现象可能在窦房结起搏活动中起到了关键作用。     

幼鼠和成年大鼠心室肌细胞起搏电流的改变

目的:运用膜片钳全细胞技术和实时定量聚合酶链式反应(PCR),探讨幼鼠和成年大鼠心室肌细胞起搏电流(If)及超极化激活的环核苷酸门控通道(HCN)亚型的改变。方法:分离3d的幼鼠和成年大鼠的心室肌细胞;测定HCN1、HCN2、HCN3和HCN4 mRNA的表达;记录If并研究其特性。结果:在新生大鼠心室肌细胞记录到If并得到电流密度-电压曲线,其激活电压约为-75mV;实时定量PCR检测HCN1、HCN2、HCN3和HCN4 mRNA在总HCN mRNA的表达中所占比例分别为0.23%±0.01%、83.58%±0.04%、0.79%±0.01%和15.44%±0.01%。在成年大鼠心室肌细胞也记录到超极化激活、并可以被4mmol/LCsCl阻断的If,其激活电压约为-115mV;HCN1、HCN2、HCN3和HCN4 mRNA在总HCN mRNA中所占比例分别为0.72%±0.02%、91.58%±0.08%、0.27%±0.02%和7.12%±0.02%。HCN2∶HCN4为(13.06±0.21)∶1。结论:随着年龄的增长,大鼠心室肌细胞HCN2所占比例增加;If值减小,激活电压变负。     

肿瘤干细胞及肝癌肿瘤干细胞

肿瘤干细胞理论认为只有存在于肿瘤中的少量干细胞性质的细胞群体对肿瘤发生和发展起着决定作用,肿瘤是由干细胞突变积累而形成的无限增殖的异常组织,这一理论的提出使人们对肿瘤发生机制的认识上升到了一个新的高度,也引起了研究者的广泛关注;肝癌是我国常见的恶性肿瘤之一,我国肝癌死亡率居世界之首,目前对肝癌的研究是我国恶性肿瘤防治的重点工作,现对当前肿瘤干细胞与肝癌肿瘤干细胞相关方面的最新研究进展作一概述。     

Fusion of Tumour Cells with Host Cells

THE A9 cell is an 8-azaguanine-resistant derivative of the L cell line¹. It lacks the enzyme inosinic acid pyrophosphorylase and is thus unable to grow in media such as HAT² in which endogenous synthesis of nucleic acid is blocked by aminopterin. The A9 line has little ability to grow progressively in vivo. Inocula of 5 × 10⁴ to 2 × 10⁶ cells produced progressive tumours in only 12% of X-irradiated newborn syngeneic C3H mice³. One of these tumours was explanted as a cell suspension into Eagle's minimal essential medium containing 15% foetal calf serum and then subcultivated in this medium with 5% foetal calf serum. At each passage, cells were inoculated into X-irradiated newborn syngeneic C3H or semi-allogeneic C3H×X F₁ mice (X designates a number of different allogeneic parents). Between 80 and 90% of the inoculated animals developed progressive tumours. The cell line was therefore designated A9HT (high take incidence). The karyotype of the A9HT line was found to be similar to that of the A9 line, but with a slightly reduced total chromosome number. The modal chromosome number of A9HT was about 53, compared with about 57 for A9 (see ref. 4). A9 and A9HT both had between 20 and 30 bi-armed chromosomes and a number of marker chromosomes in common. A detailed comparison of the karyotypes of the two lines examined by the quinacrine fluorescence technique has been made⁵. The A9HT line, like its A9 parent, lacks inosinic acid pyrophos-phorylase and is unable to grow in HAT medium.     

Dendritic Cells and Regulatory T Cells in Atherosclerosis

Although macrophages and other immune system cells, especially T cells, have been shown to play disease-promoting roles in atherosclerosis, less is known about the role of antigen presenting cells. Functional, immune stimulating dendritic cells (DCs) have recently been detected in aortic intima, the site of origin of atherosclerosis. We had compared DCs with macrophages in mice with experimental atherosclerosis, to clearly define cell types by developmental and functional criteria. This review summarizes recent advances in studies of DCs in humans and in mouse models of atherosclerosis, as well as providing a simple strategy to measure regulatory T (Treg) cells in the mouse aorta.     

Place Cells,Grid Cells,and Memory

The hippocampal system is critical for storage and retrieval of declarative memories, including memories for locations and events that take place at those locations. Spatial memories place high demands on capacity. Memories must be distinct to be recalled without interference and encoding must be fast. Recent studies have indicated that hippocampal networks allow for fast storage of large quantities of uncorrelated spatial information. The aim of the this article is to review and discuss some of this work, taking as a starting point the discovery of multiple functionally specialized cell types of the hippocampal–entorhinal circuit, such as place, grid, and border cells. We will show that grid cells provide the hippocampus with a metric, as well as a putative mechanism for decorrelation of representations, that the formation of environment-specific place maps depends on mechanisms for long-term plasticity in the hippocampus, and that long-term spatiotemporal memory storage may depend on offline consolidation processes related to sharp-wave ripple activity in the hippocampus. The multitude of representations generated through interactions between a variety of functionally specialized cell types in the entorhinal–hippocampal circuit may be at the heart of the mechanism for declarative memory formation.The scientific study of human memory started with Herman Ebbinghaus, who initiated the quantitative investigation of associative memory processes as they take place (Ebbinghaus 1885). Ebbinghaus described the conditions that influence memory formation and he determined several basic principles of encoding and recall, such as the law of frequency and the effect of time on forgetting. With Ebbinghaus, higher mental functions were brought to the laboratory. In parallel with the human learning tradition that Ebbinghaus started, a new generation of experimental psychologists described the laws of associative learning in animals. With behaviorists like Pavlov, Watson, Hull, Skinner, and Tolman, a rigorous program for identifying the laws of animal learning was initiated. By the middle of the 20th century, a language for associative learning processes had been developed, and many of the fundamental relationships between environment and behavior had been described. What was completely missing, though, was an understanding of the neural activity underlying the formation of the memory. The behaviorists had deliberately shied away from physiological explanations because of the intangible nature of neural activity at that time.Then the climate began to change. Karl Lashley had shown that lesions in the cerebral cortex had predictable effects on behavior in animals (Lashley 1929, 1950), and Donald Hebb introduced concepts and ideas to account for complex brain functions at the neural circuit level, many of which have retained a place in modern neuroscience (Hebb 1949). Both Lashley and Hebb searched for the engram, but they found no specific locus for it. A significant turning point was reached when Scoville and Milner (1957) reported severe loss of memory in an epileptic patient, patient H.M., after bilateral surgical removal of the hippocampal formation and the surrounding medial temporal lobe areas. “After operation this young man could no longer recognize the hospital staff nor find his way to the bathroom, and he seemed to recall nothing of the day-to-day events of his hospital life.” This tragic misfortune inspired decades of research on the function of the hippocampus in memory. H.M.’s memory impairment could be reproduced in memory tasks in animals and studies of H.M., as well as laboratory animals, pointed to a critical role for the hippocampus in declarative memory—memory, which, in humans, can be consciously recalled and declared, such as memories of experiences and facts (Milner et al. 1968; Mishkin 1978; Cohen and Squire 1980; Squire 1992; Corkin 2002). What was missing from these early studies, however, was a way to address the neuronal mechanisms that led information to be stored as memory.The aim of this article is to show how studies of hippocampal neuronal activity during the past few decades have brought us to a point at which a mechanistic basis of memory formation is beginning to surface. An early landmark in this series of investigations was the discovery of place cells, cells that fire selectively at one or few locations in the environment. At first, these cells seemed to be part of the animal’s instantaneous representation of location, independent of memory, but gradually, over the course of several decades, it has become clear that place cells express current as well as past and future locations. In many ways, place cells can be used as readouts of the memories that are stored in the hippocampus. More recent work has also shown that place cells are part of a wider network of spatially modulated neurons, including grid, border, and head direction cells, each with distinct roles in the representation of space and spatial memory. In this article, we shall discuss potential mechanisms by which these cell types, particularly place and grid cells, in conjunction with synaptic plasticity, may form the basis of a mammalian system for fast high-capacity declarative memory.     

体细胞重编程逆转为干细胞的研究进展

目前细胞和发育生物学上的研究成果为生物医学研究提供了广泛的前景.将完全分化的细胞重编程,不经过胚胎逆转为多能干细胞状态,这点燃了再生医学应用的新希望,这一成果从法律、道德、伦理等不同方面被人们所接受.通过体细胞克隆胚胎获得干细胞所面临的破坏胚胎的伦理限制,促使研究者去寻求将分化细胞重编程逆转为干细胞的新方法.主要论述了体细胞重编程的原理、过程及不经过胚胎逆转为多能干细胞的方法.     

Dendritic Cells: Migratory Cells that are Attractive

Dendritic cells (DC) are professional antigen presenting cells, playing an important role in the initiation of T- and T cell dependent immune responses. DC are highly mobile cells and the sequential migration of DC in and out of tissues is accompanied by phenotypical as well as functional changes instrumental to their function as sentinels of the immune system. Herein, we will review recent progress in understanding the origin of DC, their migratory behaviour and their capacity to attract and interact with lymphocytes, with emphasis on the chemokine system.     

IDS Transfer from Overexpressing Cells to IDS-Deficient Cells

Iduronate sulfatase (IDS) is responsible for mucopolysaccharidosis type II, a rare recessive X-linked lysosomal storage disease. The aim of this work was to test the ability of overexpressing cells to transfer IDS to deficient cells. In the first part of our work, IDS processing steps were compared in fibroblasts, COS cells, and lymphoblastoid cell lines and shown to be identical: the two precursor forms (76 and 90 kDa) were processed by a series of intermediate forms to the 55- and 45-kDa mature polypeptides. Then IDS transfer to IDS-deficient cells was tested either by incubation with cell-free medium of overexpressing cells or by coculture. Endocytosis and coculture experiments between transfected Lβ and deleted fibroblasts showed that IDS transfer occurred preferentially by cell-to-cell contact as IDS precursors are poorly secreted by transfected Lβ. The 76- and 62-kDa IDS polypeptides transferred to deleted fibroblasts were correctly processed to the mature 55- and 45-kDa forms. Lβ were not able to internalize the 90-kDa phosphorylated precursor forms excreted in large amounts in the medium of overexpressing fibroblasts. Enzyme transfer occurred only by cell-to-cell contact, but the precursor forms transferred in Lβ after cell-to-cell contact were not processed. This absence of maturation was probably due to a mistargeting of IDS precursors in these cells.     

Oocyte-like Cells Induced from Mouse Spermatogonial Stem Cells

ABSTRACT: BACKGROUND: During normal development primordial germ cells (PGCs) derived from the epiblast are the precursors of spermatogonia and oogonia. In culture, PGCs can be induced to dedifferentiate to pluripotent embryonic germ (EG) cells in the presence of various growth factors. Several recent studies have now demonstrated that spermatogonial stem cells (SSCs) can also revert back to pluripotency as embryonic stem (ES)-like cells under certain culture conditions. However, the potential dedifferentiation of SSCs into PGCs or the potential generation of oocytes from SSCs has not been demonstrated before. RESULTS: We report that mouse male SSCs can be converted into oocyte-like cells in culture. These SSCs-derived oocytes (SSC-Oocs) were similar in size to normal mouse mature oocytes. They expressed oocyte-specific markers and give rise to embryos through parthenogenesis. Interestingly, the Y- and X-linked testis-specific genes in these SSC-Oocs were significantly down-regulated or turned off, while oocyte-specific X-linked genes were activated. The gene expression profile appeared to switch to that of the oocyte across the X chromosome. Furthermore, these oocyte-like cells lost paternal imprinting but acquired maternal imprinting. CONCLUSIONS: Our data demonstrate that SSCs might maintain the potential to be reprogrammed into oocytes with corresponding epigenetic reversals. This study provides not only further evidence for the remarkable plasticity of SSCs but also a potential system for dissecting molecular and epigenetic regulations in germ cell fate determination and imprinting establishment during gametogenesis.